Oral immunostimulation of mammals, birds, fish and reptiles from (1-4) linked beta-d-mannuronic acid

ABSTRACT

An oral, immunostimulating material for mammals, birds, fish, and reptiles comprising an immunostimulating amount of an alginate having a M content of at least 40% and an acceptable carriers.

FIELD OF THE INVENTION

The present invention is directed to an oral, immunostimulating materialfor mammals, birds, fish, and reptiles comprising an immunostimulatingamount of an alginate having a (1-4) linked β-D-mannuronic acid contentof at least 40% and, if necessary or desired, an acceptable carrier. Thepresent invention is also directed to a method for stimulating theimmune system of a mammal, bird, fish, or reptile comprising orallyadministering the inventive material to a mammal, bird, fish, orreptile.

BACKGROUND OF THE INVENTION

Alginates are isolated from marine brown algae. Alginate is alsoproduced in some soil bacteria such as Azotobacter vinelandii andAzotobacter crococcum and several different Pseudomonas sp. Brown algaare however generally the source of commercially available alginates.

Alginates are salts of alginic acid, a linear, hetero polysaccharideconsisting of (1-4) linked β-D-mannuronic acid, designated herein as M,and α-L-guluronic acid, designated herein as G. These two uronic acidshave the following formulae:

The polymers exist as homopolymer sequences of mannuronic acid, calledM-blocks, homopolymer sequences of guluronic acid called G-blocks, andmixed sequences of mannuronic and guluronic acid units, designatedMG-blocks or alternating blocks. The following scheme represent anillustration of the structure of alginates:MMMMMMMGGGGGGGGMGMGMGMGMGGGGGGGGM  M-block G-block MG-block G-block

Alginates usually contain all three types of blocks and a block mostlyconsists of three to thirty monomer units. The distribution of theblocks depends on the type of algae from which the alginate is isolated,as well as on the age and part of the plant, for example alginate fromthe stem may have a different sequence and block composition to alginateisolated from the leaves. The time of year at which the algae areharvested also affects the block composition and sequence. According tothe common knowledge, the highest G-content can be found in the stem ofold L. hyperborea. The leaf of the same species has a somewhat lowerG-content and shorter G-blocks, but the content is still higher thanmost other species. Commercially available alginates usually have aG-content of 25%-70%.

Alginates are known to be used in foodstuffs and in pharmaceutical,dental, cosmetic and other industrial products. The most commonindustrial applications are based on their hydrocolloidal andpolyelectrolytic nature, which forms the basis for the gel-forming,thickening, stabilizing, swelling and viscosity-providing properties.

Alginates that are rich in M content have also been shown to possessimmunostimulating activity useful as vaccine adjuvants and wound-healingcompositions as described in U.S. Pat. No. 5,169,840.

SUMMARY OF THE INVENTION

The present invention is directed to an oral, immunostimulating materialfor mammals, birds, fish, and reptiles comprising an immunostimulatingamount of an alginate having a M content of at least 40% and, ifnecessary or desired, an acceptable carrier.

The present invention is also directed to a method of stimulating theimmune system of a mammal, bird, fish, and reptile comprising orallyadministering an immunostimulating amount of an immunostimulatingingestible material to said mammal, bird, fish, and reptile, whereinsaid material comprises an alginate having a M content of at least 40%and, if necessary or desired, an acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

The following abbreviations are used in the figures; Durvillea waterextract=DWE, Durvillea standard extract=Std. DA and Lessonia standardextract=Std.LN.

FIG. 1 is a graph showing the weight increase of pigs given feedcontaining alginates of the invention over a twelve week period.

FIG. 2 is a graph showing the serum level of total white blood cells inthe pigs tested with alginates of the invention as compared to acontrol.

FIG. 3 is a graph showing the serum level of monocytes in the pigstested with the alginates of the invention as compared to a control.

FIG. 4 is a graph showing the serum level of lymphocytes in the pigstested with the alginates of the invention as compared to a control.

FIG. 5 shows the level of phagocytosis measured using blood from pigsfed Durvillea water extract as compared to a control.

FIG. 6 shows the oxidative burst measured using blood from pigs fed theDurvillea water extract as compared to a control.

FIG. 7 shows the immune response to an injected Human Serum Albuminexperimental vaccine for pigs fed Durvillea standard extract, Durvilleawater extract and Lessonia standard extract as compared to a control.

FIG. 8 shows the specific growth rate (% growth per day) of spottedwolffish fed with oral feed containing Durvillea water extract over asixty day period viz-a-viz the specific growth rate of a control group.

DETAILED DESCRIPTION OF THE INVENTION

Alginates having an M content of at least 40% are utilized as oralimmunostimulators in the present invention for mammals, birds, fish, andreptiles. More specifically, alginates having an M content of 50% to 70%(such as derived from Lessonia, Durvillea, and Laminaria); 70% to 80%(such as derived from Durvillea) and 80% to 99.9% (such as derived frombacteria and water extracts of alginates like Durvillea as prepared, forexample, in accordance with the examples below) may be used. Thesealginates stimulate the immune response of mammals, birds, fish, andreptiles against disease or trauma caused by cellular attack by foreignbodies and physical injury of cells. Included among foreign bodies aremicroorganisms, particulate matter, chemical agents and the like.Included among physical injuries are mechanical wounds such asabrasions, lacertions, contusions, wounds and the like.

The oral immunostimulating material and method of the present inventionutilize an immunostimulating amount of the alginate having an M contentof at least 40%. The immunostimulating amount can vary depending on thesubject that is to ingest the immunostimulating material and the levelof the immunostimulation that is needed. For example, without beinglimiting, for the first 60 days of a fish's life, orally administer 2-20mg alginate/fish. Of course, more matured fish will require largeramounts.

The oral immunostimulating material containing the alginate having an Mcontent of at least 40% may be pharmaceutical, veterinary ornutraceutical solid dosage forms such as tablets, caplets, capsules,etc, or as a powder or liquid formulation. It may also be any type ofsolid or liquid food for mammalian, avian, ichthyic, or reptilianconsumption such as pet food. It may also be a solid, semi-solid orliquid nutritional supplement such as food bars, drinks, etc.

Acceptable carriers can be any of those conventionally used inpharmaceutical, veterinary and nutraceutical liquid or solid dosageforms, liquid, solid and semi-solid foods and liquid and solidnutritional supplements.

It has also been found, as demonstrated below in the Examples, that theimmunostimulating activity of the alginates of the invention stimulatethe weight gain for a mammal (as demonstrated below in pigs (see FIG.1)) and a fish (as demonstrated below in spotted wolffish (see FIG. 8 ))ingesting the alginate as compared to a control. This aspect isparticularly useful and desirable when such alginates are orally givento young mammals. As such, the present invention is also directed to amethod for stimulating weight gain in mammals, birds, fish, and reptilesthrough immunostimulation comprising orally administering animmunostimulating amount of an immunostimulating material to the mammal,bird, fish, or reptile, wherein the material comprises an alginatehaving a M content of at least 40% and, if necessary or desired, anacceptable carrier. More specifically, this method also comprisesadministration of an alginate having an M content of 50% to 70%, 70% to80% or 80% to 99.9%. The immunostimulating material may be any of thoseset forth above.

The oral immunostimulating material of this invention can containalginate having an M content of at least 40% that is syntheticallyderived or isolated from either alginate-producing bacterial species orseaweed sources.

Alginates having an M content of at least 40% can be obtained fromseaweeds by many processes known in the art. The starting material ofthe alginate having the required M content is algae or seaweed, inparticular, brown algae which is generally treated with formaldehyde inorder to fixate the phenols and preserve the algae. Further, the algaecan be washed with acid to remove the highly viscous laminaran andfucoglycans. Preferably, they can also be treated with alkali to reducethe content of pyrogens. It is understood that the algae can bepre-treated in any known manner. Commercially available alginates, mostpreferably, dried and milled algae of the species Durvillea can be used,but also fresh, whole or unmilled algae from Durvillea, Laminaria,Lessonia, Ecklonia, Macrocystis, or Ascophyllum are suitable as startingmaterials.

Processes for producing such alginates are set forth, for example, byGreen in U.S. Pat. No. 2,036,934 and Le Gloahec in U.S. Pat. No.2,128,551 and such processes are incorporated herein by reference. Othermethods for obtaining alginates useful in the invention are providedhereinbelow in the examples. For example, alginates of the invention mayalso be prepared using a water extraction process by mixing an alginatesource having a high M content with water in a ratio of 1:3 to 1:20 in aswelling step where the pH is kept above about 2.3 at a temperatureabove 20° C. for at least 30 minutes, and isolating the solubilisedalginate fraction from the solid material by filtration. An alginatehaving the required M content can be recovered from the solution byprecipitation with acid, salt or alcohol.

The Examples set out below include representative examples of aspects ofthe present invention. The Examples are not meant to limit the scope ofthe invention but rather serve exemplary purposes. Unless indicatedotherwise, all parts, percentages and the like are by weight.

EXAMPLE 1

Starting raw material from different Durvillea species; D.potatorum(milled), sample 1, and D.antarctica (not milled), sample 2, were addedwater in the amounts set forth in the table below, and stirred by handfrom time to time, at a temperature of 55° C. for 3.5 hours. Afterstorage at ambient temperature over the night, the algae were extracteda second time at 55° C. for 1 hour, then 2.5 ml formaldehyde was addedand the extraction continued for 1 hour. Weight Water Time TemperatureFormaldehyde Sample [gram] [ml] [hours] [° C.] [ml] pH 1 50.0 500 5.5 552.5 5.9 2 40.0 500 5.5 55 2.5 6.9

The suspension was then sieved on a 60-mesh filter and washed 2 timeswith an excess of water. The solution was then filtrated with filteraids on a vacuum funnel and thereafter on a pre filter of glass filter.The solution was then allowed to cool to 10° C. and was then added NaClto a 0.5% concentration. Thereafter drops of dilute 5.5 M hydrochloricacid were added by stirring with magnet to a pH 1.8. A white precipitatewas formed. The suspension was, after being kept at 10° C. for 30minutes, sieved on a 120 mesh-filter cloth and pressed by hand resultingin pasty, yellow mass, that turned to fine fiber after pressing. All theacid material was transferred to a 250 ml vessel and added water to 200ml, before neutralized to pH 7, with solid soda ash under magneticstirring. The solution was once again filtrated on a 0.8 micron filtermembrane of cellulose nitrate. The filtrate was cooled to 10° C. andprecipitated with isopropyl alcohol in the ratio 1:1. The fibers formedwere washed once with 70 volume % isopropyl alcohol and then a secondtime with 100 volume % isopropyl alcohol. The fibers were drawn out witha pincer and then freeze-dried. Results are given in the followingtable. TABLE Product Weight Yield % Alginate Sample Alga [gram] [gram](hot water extracted) 1 D. potatorum 50 1.08 2.1 2 D. antarctica 40 1.594.0

Analysis of product Intrinsic Molecule viscosity weight % Mannuronicacid Sample Alga dL/g Dalton/g NIR model ALGLN2D 1 D. potatorum 2.7  44009 82 2 D. antarctica 7.0 118 892 88

Block-distribution of product measured on NMR 400 Hz Sample Alga M G MMGG GM/MG 1 D. potatorum 85.9 14.1 76.3 4.5 9.6 1 D. antarctica 90.9 9.184.8 3.0 6.1

The following table shows yields of M prepared from otherseaweed-samples pursuant to the process in this example. Mannuronic DryMatter Yield Acid Alga/Seaweed Form % w/w % % Ascophyllum nodosum,spring Whole, cut 20 0.035 90 Durvillea antarctica, Chile, 1996 Unmilled85 4 91 Durvillea antarctica, Chile, 1996 Milled 85 6 89 Durvilleaantarctica, Chile, 1998 Whole 85 1.3 87 Durvillea antarctica, Chile,2000. Milled 85 2.5 91 Durvillea potatorum, Tasmania, 1997 Milled 85 2.186 Lessonia trabeculata, Chile, 1996 Milled 85 0.125 NA Lessonianigrescens, Chile, 1995 Milled 85 — NA Laminaria hyperborea, leavesFresh, cut 18 — NA Saragassum, Tanzania, aug, 1991. Milled 85 — NAMacrocysts pyrifera, Chile, 1994 Milled 85 — NA Laminaria japonica,Japan, 1988 Whole, cut 85 0.2 NA Fucus spiralis, summer, 1994 Whole, cut15 0.026 91

EXAMPLE 2

A sample from August 1996 of Durvillea antarctica milled to particleslarger than 70 mesh was used as the starting material. 30 gram of driedalgae was weighted in a vessel. 100 ml 0.2 M HCl was added and thematerial was diluted with water to 500 ml. After a few minutes ofstirring the pH increased to >2.3 and acid was added to keep pH lessthan 2.3, (pH 1.8). After 2 minutes 2.5 ml of 0.2M HCl was added. Thematerial swelled very little when the pH was kept constant under pH 2.3at pH 1.8, compared to swelling with pure water. After swelling for 1hour, the material was sieved on 60 mesh filtration cloth, pressed byhand and transferred to a vessel. The resulting material was then added500 ml of water and 50 ml soda ash/sodium hydroxide solution andextracted at 55° C. for 1 hour. The material swelled very quickly andbecame thick like a paste or pulp. It was stored by ambient temperatureuntil next day. Then the material was further extracted for 1 hour at55° C. and then milled on a mixer unit. Total mass was weighed to 549gram. 150 gram of material was diluted with 700 gram of water understirring. The solution was then filtrated on filter paper after additionof filter aids by means of vacuum from water suction. The amount offiltrate was measured to 564 gram and was cooled to 10° C. It was addedsodium chloride to 0.5% and pH was adjusted to 1.6 with drops of dilutedhydrochloric acid (1:1). A soft precipitate was formed. The material wasthen sieved on a 120-mesh filtration cloth and pressed carefully byhand. The material was then suspended with water and diluted to a volumeof about 200 ml at a temperature of 20° C. The pH of the solution wasneutralized to 7 with solid soda ash powder by using a magnetic stirrer.The solution was precipitated with equal parts of isopropyl alcoholsolution by stirring with a glass rod. The precipitated fiber was washedonce with 70 volume % isopropyl alcohol solution. Then washed again withpure 100 volume % isopropyl alcohol. After sieving and pressing on a120-mesh filtration cloth, the fibers were drawn out by with pincer andthen freeze-dried overnight with vacuum. The yield was weighed to 1.04gram of alginate, corresponding to 3.6 weight % from the DurvilleaAntarctica starting material. The content of M was 70% and theblock-distribution of the alginate measured by NMR as follows. AnalysisM G GG MM GM/MG NIR (algln2d) 77 23 — — — NMR 78.7 21 10.9 68.3 10.4

EXAMPLE 3

It is possible to further increase the content of mannuronic acid byaddition of salt in the pre-extraction step. 20 gram Durvilleaantarctica (milled coarse particles>70 mesh) algae from Chile August1996 was added 500 ml water and a certain amount of NaCl and wasextracted under stirring on a Jar test machine, at stirring speed 140rpm for 2 hours at a temperature of 20° C. The salt was added to aconcentration in the solution as set forth in the following table. TABLEDurvillea Extraction Antarctica time, Sample [gram] Water [ml] NaClconc. [hours] Remarks A 20 500 0 2 B 20 500 0.2% 2 C 20 500 0.5% 2 D 20500 1.0% 2 E 20 500 2.0% 2 F 20 500 3.0% 2 G 20 500 3.4% 2 H 20 500 — 2Seawater

The material was then sieved on a 400 mesh filtration cloth and pressedby hand. The sieved solution was weighed and pH measured as given in thefollowing table. TABLE NaCl Amount sieved Sample conc. [gram] pH RemarksA 0 377 6.3 B 0.2% 371 6.0 C 0.5% 389 6.0 D 1.0% 397 5.9 E 2.0% 417 5.8F 3.0% 444 5.8 G 3.4% 455 5.0 H — 421 6.4 Seawater

The sieved solution was then filtrated in vacuo (water suction pump) ona funnel with filter paper. The viscosity of the filtrated solution wasmeasured on a glass tube and the results are given in the followingtable. TABLE Amount of Time Viscosity NaCl filtrate Measured, calculatedSample conc. [gram] [seconds] [cps] A 0 277 18.7 11.6 B 0.2% 286 16.09.9 C 0.5% 319 13.8 8.6 D 1.0% 330 12.2 7.6 E 2.0% 408 7.5 4.7 F 3.0%438 4.9 3.0 G 3.4% 445 — 2.6 H — 402 — 2.9

The filtrate was cooled to below 15° C. and each of the samples wereadded drops of 5.5 M hydrochloric acid until the pH reached 1.8-2.0,under stirring with a magnetic stirrer. A fiber shaped precipitate wasformed. The precipitate was then sieved on a 400-mesh filtration clothand pressed by hand. The alginic acid was then diluted with water andneutralized with solid soda ash to pH 6-7 under stirring untilcompletely solved. The solution was then cooled and precipitated withequal parts of isopropyl alcohol. Thereafter washed with 70 volume %isopropyl alcohol, and repeatedly washed with pure 100 volume %isopropyl alcohol. The precipitated fiber was pulled out with a pincerand transferred to a vessel and freeze dried overnight in vacuum. Theresults are shown in the following table, wherein the amount of yieldwas calculated assuming that no alginate was lost and that all thealginate is solved in the water added. TABLE Amount M-block. alginateAmount % % NIR NaCl precipitated alginate yield yield Model Sample conc.[gram] [g/l] 100% real ALGLN2D A 0 1.26 3.34 8.4 4.6   88% B 0.2% 1.033.60 9.0 5.1 C 0.5% 1.1 3.48 8.7 5.6 D 1.0% 1.14 3.45 8.6 5.7 E 2.0%1.20 2.87 7.2 5.9 F  30% 0.80 1.80 4.5 3.9 G 3.4% 0.54 1.21 3.0 2.791.8% H Seawater 0.53 1.32 3.3 2.7 95.8%

EXAMPLE 4

The content of mannuronic acid in the separate fraction was furtherincreased by addition of CaCl₂. The starting material was D.antarcticafrom Chile that was milled to coarse particles>70 mesh. The amounts andconditions of the pre-extraction step are set out in the followingtable. The pre-extraction was carried out under stirring on Jar testerwith about 140 rpm. TABLE Weight Water Time Temperature Calcium Sample[gram] [ml] [hours] [° C.] pH chloride [N] A 20.0 500 2 25 6.08 0.01 B20.0 500 2 25 5.85 0.1 C 20.0 498 2 25 5.8 0.03 D 20.0 496 2 25 5.7 0.06

The material was then sieved on a 400-mesh filter and pressed by hand.The solution was then heated to about 30° C. and filtrated with paper ona vacuum suction flask. TABLE Calcium Sieved sol. Filtrated chloride 400mesh solution Sample [N] [gram] [gram] A 0.01 404 397 B 0.1 462 457 C0.03 423 398 D 0.06 440 420

The solution was then cooled to 10° C. and added sodium chloride to0.5%. Then drops of 5.5 M HCl was added, with carefully magneticstirring until pH 1.8. A white precipitate was formed. The materialsuspension was then stored for 30 minutes and sieved on 400-meshfiltration cloth and pressed carefully by hand. The material was a pastyyellow mass, which turned to fine bright fibers after pressing. The acidmaterial was then transferred to a 250 ml vessel and added water to 200ml and then neutralized pH 7 with solid soda ash under magneticstirring. The filtrate was then cooled to 10° C. and precipitated onstirring with 100 volume % isopropyl alcohol in a ratio 1:1. Largefibers were precipitated. The fibers were washed twice with 70 volume %isopropyl alcohol and finally with 100 volume % isopropyl alcohol. Thefibers were then pulled out with a pincer, and thereafter freeze-driedovernight under vacuum. The results are given in tables 9 and 10 whichshow the yields and the increase in content of mannuronic acid in thealginate with more than 80% M from maximum 91%, when salt not was added,to a maximum of 95%, when salt was added to the pre-extraction step.TABLE Calcium Yield Chloride Yield alginate Alginate Yield Yield Sample[N] [gram] [g/l] 100% % real % A 0.01 1.29 3.19 8.0 6.5 B 0.1 — 0 0 0 C0.03 0.52 1.31 3.28 2.6 D 0.06 0.19 0.45 0.33 0.9

TABLE Calcium % Mannuronic Chloride acid % M % G % MM % GM/MG % GGSample [N] NIR model LND 2 NMR NMR NMR NMR NMR A 0.01  98.7 89.0 11.082.0 8.0 3.0 B 0.1  — — — — — — C 0.03 101.6 92.0  8.0 86.0 6.0 1.6 D0.06 102.5 95.0  5.0 90.0 5.0 0   E water only 88.7 11.3 80.4 8.3 3.0

EXAMPLE 5

A total of forty eight cross breed [(NorwegianLandrace×Yorkshire)×Norwegian Landrace] 35 to 38 days old weanling pigsfrom six litters with an average initial weight of 13.07 kg (STD 1.98)were divided into four groups for an experimental feeding study andplaced in an environmentally controlled nursery. Initially pigs werehoused at four pigs per 2.5 m by 2.5 m pen. After six weeks of feeding,the pigs were transferred to 15 square meter pens with individualfeeding stalls with six pigs per pen. Pigs were allowed access to feedand water ad libitum. Pigs were individually weighed on a weekly basis.Pigs were allowed a 4 day preliminary period of adjustment to the pensand the standard commercial weaningly pig feed. The commercial feed (13%moisture content) was fortified with vitamins and minerals required forgrowth and had a raw protein content of about 18-19%, a digestibleprotein content of 14-15%, a starch content of 40-50%, and a raw fatcontent of about 2.6%.

After the initial adjustment to commercial feed, four groups of eachtwelve pigs were given a different diet for ten weeks. The final twoweeks of the study the pigs were all given the commercial feed. Pigs inthe control group were fed the standard commercial feed while pigs inthe other three groups were fed the same commercial feed which had beenblended with 1.25% (w/w) of alginate. The three alginates tested were 1.Durvillea antarctica water extract (89% M), 2. Durvillea antarcticastandard extract (63% M) and 3. Lessonia nigrescens standard extract(55% M). These alginate powders as characterized by intrinsic viscosityand ¹H NMR spectroscopy measurements are further described as follows:

Durvillea antarctica standard extract had an intrinsic viscosity of 7.0g/dl which corresponds to an estimated weight average molecular weightof 210,000 Daltons using the Mark Houwink equation. This Durvillaalginate sample had a mole fraction content for mannuronate (M) units of0.63 and for guluronate (G) units of 0.37. The fractional content of theM to M linkages, F(MM), was 0.44. The fractional content for the G to Glinkages, F(GG), was 0.18. The fractional content of the M to G linkage,F(MG), which is equal to the fractional content of the G to M linkageF(GM), was 0.19.

Durvillea antarctica water extract or Durvillea water extract, obtainedby selective precipitation of an alginate fraction having an increasedthe M content, had an intrinsic viscosity of 4.7 g/dl which correspondsto an estimated weight average molecular weight of 85,000 Daltons usingthe Mark Houwink equation. This Durvillea water extract alginate samplehad a mole fraction content for mannuronate (M) units of 0.88 and forguluronate (G) units of 0.12. The fractional content for the M to Mlinkages, F(MM), was 0.80. The fractional content for the G to Glinkages, F(GG), was 0.04. The fraction content of the M to G linkage,F(MG), which is equal to the fractional content of the G to M linkageF(GM), was 0.08.

Lessonia nigrescens standard extract had an intrinsic viscosity of 13.4g/dl which corresponds to an estimated weight average molecular weightof 370,000 Daltons using the Mark Houwink equation. This Lessoniaalginate sample had a mole traction content for mannuronate (M) units of0.55 and for guluronate (G) units of 0.45. The fractional content of theM to M linkages, F(MM), was 0.35. The fractional content for the G to Glinkages, F(GG), was 0.25. The fractional content of the M to G linkage,F(MG), which is equal to the fractional content of the G to M linkageF(GM), was 0.20.

The alginate intrinsic viscosity was determined using the methoddisclosed in Alginates as immobilization materials—A study of somemolecular and functional properties (Martinsen, Anita) Thesis;NTH—University of Trondheim, 1990. The weight average molecular weightwas estimated based on the intrinsic viscosity data and the Mark Howinkequation. The monomer composition and sequential arrangement wereanalyzed by ¹H-NMR spectroscopy on a Brucker 400 WM spectrometer asdescribed by Grasdalen et al, “A NMR studie of composition and sequenceof uronate residues in alginate”, in Carbohydrate research 1979; 68:23and H. Grasdalen “High Field ¹H NMR spectroscopy of alginate: sequentialstructure and linkage conformation” in Carbohydrate Research, 1983;118:255.

After two weeks of feeding, the pigs were immunized (at 7 weeks of age)and they were given a booster injection four weeks later (at 11 weeks ofage). All pigs were immunized intramuscularly with 0.5 ml of a mixtureof 0.25 ml Pneumabort K® (102BY0002, Fort Dodge Laboratories) containingequine herpes virus 1 (EHV) and 0.25 ml Human serum albumin (HSA)(200μg/ml) (Sigma), subcutaneously with 0.5 ml Prevacun F® (027021E, HoechstRoussel Vet, Germany) containing influenza virus A/Equi 1, A/Equi 2/M,A/Equi 2/F (EIV) and 1.0 ml Diphteri/Tetanus vaccine® (DT9169a1, SBLVaccin AB, Stockholm) containing diphtheria toxoid (30 Lf/ml) andtetanus toxoid (7.5 Lf/ml).

Specific antibodies against Human serum albumin (HSA) (Sigma ChemicalIndustries, USA) and Diphteriae toxoid (DIF) (National Institute ofPublic Health) were measured by a passive haemagglutination test(Avrameas et al 1969). The lowest dilution tested was 1:8. Sera thatshowed no inhibition were given a titre of 4 for use in the statisticalcalculation. The antibody titre values were log₂-transformed tonormalize the distribution

Blood samples were collected at two week intervals for serology,haematology, and for functional characterization of phagocytes andlymphocytes. Serum was isolated and stored at −20° C. until processed.Stabilized (heparinized and EDTA) blood samples were collected in themorning and analysed immediately. The blood samples were analysed(Technicon H-1) at the Central Clinical Laboratory at The NorwegianSchool of Veterinary Science, Oslo. The total leukocyte count(WBC×10⁹/L), number (×10⁹/L) of monocytes, neutrophils and eosinophilswere measured electronically and the number of lymphocytes and therelative number (%) of lymphocytes, monocytes, neutrophils andeosinophils were estimated. The total erythrocyte counts (RBC×10¹²/L),the mean cell volume (MCV fL) and haemoglobin (HGB g/L) were measuredand the hematocrit (HCT L/L) was estimated.

The phagocytic activity of granulocytes in the blood was assayed usingPhagotest® (Orpegen Pharma, Heidelberg) following the operator's manual.Heparinized (15 IU/ml) whole blood was mixed and aliquoted (100 μl) onthe bottom of a 5 ml vial (Falcon) and incubated 10 minutes on icebefore adding 20 μl precooled stabilized and opsonized FITC-labelled E.coli suspension (test kit). All vials were shaken and the test sampleswere incubated for 10 minutes at 37° C. in a water bath, whereas thecontrol samples remained on ice. All samples were then simultaneouslyplaced on ice in order to stop phagocytosis and 100 μl of ice coldQuenching solution was added to each samples and mixed on a vortexmixer. The samples were then added 3 ml Washing solution, mixed and thecells were spun down (250×g, 5 min, 4° C.). The washing procedure werethen repeated before adding 200 μl of DNA staining solution The sampleswere mixed and incubated 10 minutes on ice, and the cells were analysedby flow cytometry (FACScan™, LYSIS™ software) using the blue-greenexcitation light (488 nm). The percentage of cells having preformedphagocytosis was analysed.

The evaluation of oxidative burst activity was performed by flowcytometry using Phagoburst® (Orpegen Pharma, Heidelberg) following theoperator's manual. Heparinized (15 IU/ml) whole blood was mixed andaliquoted (100 μl) on the bottom of a 5 ml vial (Falcon) and incubated10 minutes on ice before adding 20 μl precooled stabilized and opsonizedE. coli suspension (test kit). Three control vials were included foreach animal tested; one tube was added 20 μl of Washing solution(negative control), one tube was added 20 μl of the chemotactic peptidefMLP working solution (“low control”) and one tube was added 20 μl ofphorbol 12-myristate 13-acetate (PMA) working solution (“high control”).All vials were mixed and the test samples were incubated for 10 minutesat 37° C. in a water bath. All samples were then added 20 μl ofSubstrate solution, mixed thoroughly and incubated for another 10minutes at 37° C. in a water bath. All samples were then simultaneouslytaken out of the water bath and the whole blood was lysed and fixed with2 ml prewarmed Lysing solution, mixed and incubated for 20 min at roomtemperature. The samples were spun down (250×g, 5 min, 4° C.) and washedonce by adding 3 ml Washing solution (250×g, 5 min, 4° C.). Thesupernatant was decanted and 200 μl of DNA staining solution was added,the samples were mixed and incubated 10 minutes on ice (lightprotected). The cells were analysed by flow cytometry (FACScan™, LYSIS™software) using the blue-green excitation light (488 nm). The percentageof cells having produced reactive oxygen metabolites was analysed aswell as their mean fluorescence intensity.

FIG. 1 shows mean weight for the four groups of pigs as a function offeeding time. A significant weight increase was found for pigs in theDurvillea water extract group and Lessonia group compared to the controlgroup. The weight difference is deemed statistically significant afterweek 3, however FIG. 1 shows a trend in weight gain consistent with theDurvillea water extract group and the Lessonia group increasing inweight more rapidly than the control group after week 1. While theactual weight differential between the Durvillea group and the controlgroup is not as dramatic as that seen for the Durvillea water extractgroup and Lessonia group, it should be noted that the mean weight of theDurvillea group was consistently higher than the control. The individualpigs in the control group had a higher variability in weight compared toindividuals in the three groups which had alginate-containing feed whichsuggests that some individuals in the control group had a reduced immuneresponse since they were less able to cope with trauma and/or stress.The hormone Cortisol in pigs partitions nutrients away from muscle andadipose tissue to allow the animal to cope with disease stress.

FIGS. 2 through 4 show data obtained by analyzing the blood for totalwhite blood cells, monocytes and lymphocytes. While not statisticallysignificant, FIG. 2 indicates that the total white blood cells forDurvillea water extract group is higher than the control group. Theincrease white blood cell count observed for the Durvillea water extractgroup is due to significant increases in both monocytes (FIG. 3) andlymphocytes (FIG. 4) after 6 and 10 weeks, respectively, for theDurvillea water extract group compared to the control group. Thelymphocytes for the Lessonia group show a delayed increase (week 10)compared to the Durvillea water extract group. The blood taken from theDurvillea water extract group (and control group) were also assayed forphagocytic activity, and significant increases were measured for theDurvillea water extract group compared to the control group at week 4 asseen in FIG. 5. The oxidative burst data shown in FIG. 6 furthersupports the higher level phagocytic response for the pigs fed theDurvillea water extract compared to the control at both week 4 and week8. The sustained increased oxidative burst at week 4 and week 8 for theDurvillea water extract group compared to the control group issignificant since the underlying chemical processes associated withrespiratory burst are necessary to kill some types of bacteria andindicates improved effectiveness of phagocytosis.

FIG. 7 indicates the immune response to vaccination. The immunoresponseto injection of Human Serum Albumin experimental vaccine (FIG. 7) showsa significant increase at week 8 for the Durvillea water extract groupand the Lessonia group which indicates an improved immune responsecompared to the control.

EXAMPLE 6

Durvillea water extract alginate (as described in Example 5) wasdissolved in water, and after a short sonication treatment, the solutioncontaining the dissolved alginate at a concentration of 4.2 gram ofalginate per liter was sprayed onto commercial feed particles(Skretting/Nutreco, Dirdal, Norge) to give 0.02% and 0.06% alginate (%by weight) on the feed. The feed particles were air-dried for two daysbefore oral administration to newly hatched spotted wolffish (Anarhichasminor) fish fry. This Durvillea antarctica water extract had anintrinsic viscosity of 8.3 dl/g which corresponds to an estimated weightaverage molecular weigh of 160000 Daltons using the Mark Houwinkequation. This Durvillea water extract alginate sample had a molefraction content for mannuronate (M) units of 0.89 and for guluronate(G) units of 0.11. The fractional content for the M to M linkages,F(MM), was 0.81. The fractional content for the G to G linkages, F(GG),was 0.03. The fraction content of the M to G linkage, F(MG), which isequal to the fractional content of the G to M linkage F(GM), was 0.09.

Three replicate groups consisting of 50 fry were used for each conditionin a 60 day feeding study with continuous feeding of the fish. Theaverage weight per fish was approximately 0.4 g at the start of thefeeding study. The fry were weighed every 10 days (wet weight) duringthe study. The test conditions included three different feeds: control(0% alginate), 0.02% alginate and 0.06% alginate. An additional group offish that fed the control feed were bath treated twice during the sixtyday feeding period by transferring the fish into a bath containing 0.1 gof alginate per liter of seawater with a treatment for 12-24 h pertreatment cycle then in the feeding study.

As shown in FIG. 8, the fish that were fed alginate-containing feedshowed a higher specific rate of growth of 5.197% growth per day (for0.02% alginate) and 5.247% growth per day (for 0.06% alginate) comparedto fish that were fed the control feed without added alginate (4.857%growth per day) over the 60 day growth period. The fish that wereintermittently bath treated twice during the 60 day study showed nosignificant different in their specific rate of growth compared to thecontrol group (4.745% growth per day vs. 4.857% growth per day,respectively). The specific growth rate in FIG. 8 was calculated as thepercentage daily growth according to the following formula: 100% times[ln (weight at the end of the study) minus ln (weight at day zero of thestudy)] divided by the number days in the study (note: ln is naturallog).

EXAMPLE 7

Durvillea water extract alginate (as described in Example 5) wasdissolved in water to give a concentration of alginate of 3%. ThisDurvillea antarctica water extract had an intrinsic viscosity of 4.7dl/g which corresponds to an estimated weight average molecular weigh of85000 Daltons using the Mark Houwink equation. This Durvillea waterextract alginate sample had a mole fraction content for mannuronate (M)units of 0.88 and for guluronate (G) units of 0.12. The fractionalcontent for the M to M linkages, F(MM), was 0.80. The fractional contentfor the G to G linkages, F(GG), was 0.04. The fraction content of the Mto G linkage, F(MG), which is equal to the fractional content of the Gto M linkage F(GM), was 0.08.

Portions of approximately 1200 grams of commercial feed (BiomarEcoweaner EMB 16244) were spray coated with different amounts of thealginate solution in a bench top fluidised bed reactor under thefollowing conditions: Air flow sufficient to fluidise the particles,40-50 degrees Celsius, and flow of alginate solution of 60 ml per hour.Dry feed particles were recovered with different concentrations of thealginate (0.01%, 0.06% and 0.10%) as calculated based on the alginateconcentration and amount of alginate solution coated per weight of drypowder originally charged. For the lowest concentration an alginatesolution diluted to 0.5% was used instead. The control sample was usedwithout further treatment. Feed with particle size of both 0.6 mm and1.0 mm was used for the early and late feeding period respectively. Thecoated and uncoated feed particles were visually inspected in amicroscope, and no difference could be seen in appearance or in particlesize. The dry matter content immediately after processing was slightlyhigher in the coated particles (95%) compared to the uncoated (90%).

To measure the loss of alginate from the feed particles, Durvilleaantarctica water extract alginate was isotope labelled with subsequentmeasurements of released alginate from coated particles in seawater. 100mg alginate was dissolved in 50 ml of 0.05 M borate buffer at pH 8.0.100 mg of p-OH methyl benzimidate (MPHBIM) was added and the solutionwas shaken in 24 hours at 37 degrees Celsius. The solution was dialysedfor 2 days against distilled water containing 1% sodium azide. Furtherdialysis in 3 days against polyethylene glycol with molecular weight of6000 was performed to make the alginate solution more concentrated. Alldialysis solutions were exchanged twice per day. The solution oflabelled alginate was filtered through a 0.45-micron filter beforefurther labelling with ¹²⁵Iodine (¹²⁵I). 50 micro litres of iodogen(1,3,4,6-tetrachloro-3alfa, 6alfa-diphenylglycoluril) was added to anEllermann tube. When the solution was evaporated from the tube, it wasflushed 5 times with water and dried with compressed air. The tube wasadded 50 micro litres of phosphate buffer solution, 4 micro litres of¹²⁵I₂, and 2 ml of the MPHBIM labelled alginate solution. The reactionwas stopped after 1 hour by adding 100 micro litres of 0.1 M sodiumbisulphite. Non-reacted isotope compounds were removed with gelchromatography using a Sephadex G-25 material. The non-reactor compoundsand labelled alginate were identified using a gamma counter. Feedparticles were coated with the ¹²⁵I₂ ⁻MPHBIM-labelled alginate as inexample 6. The coated feed particles were shaken in seawater for 5minutes, and after sedimentation of samples of the water were taken foranalysis in gamma counter. Six parallels were analysed and the loss ofalginate was measured from 5.2% to 8.0%. The experimental loss in thefeeding trials is thus considered to be very low since the larvaenormally consume the feed in seconds.

Newly hatched larvae of spotted wolffish (ca. 0.3 gram) were placed in12 containers, about 80 in each. The containers measured 40×20 cm, andwas individually supplied with continuously running water in one end sothat a continuously water current through the container was obtained.Water temperature was 8 degrees Celsius throughout the experiment.Non-consumed feed and faeces was continuously removed. Three parallelcontainers were given feed with each concentration and three containerswere given the control feed. Feed was given several times (approximatelyevery hour) during the daytime to ensure feed was not a limiting factor.Every 10 days the weight of 30 larvae was measured. The experimentlasted for 60 days. Bars marked with an “*” indicates a significantdifference (p<0.05). Results of specific growth rate are given in FIG.9.

EXAMPLE 8

Durvillea water extract alginate (as described in Example 5) wasdissolved in water, and portions of commercial feed (Biomar EcoweanerEMB 16244) were coated with different amounts of the alginate solutionin a bench top fluidised bed reactor. Dry feed particles were recoveredwith different concentrations of the alginate (0.01%, 0.06% and 0.10%)as in Example 7.

Cod larvae were acclimatized for 10 days in total before start ofexperiment. During this initial 10-day period, the larvae were all fedthe control feed and reached a weight of 0.5-1.0 g. 3 days before thestart of the experiment the larvae were divided into 12 round containers(40 cm diameter and height of 60 cm) of 30 litres with about 70 larvaein each container. Every container was equipped with plastic sieves inthe bottom. The water volume varied between 15 and 20 litre throughoutthe experiment. In the same period the water temperature followed thenatural sea temperature and increased from 8-12 degrees Celsius. Thecontainers had individual water supply of about 1.5 litres per minuteand constant light exposure. Non-consumed feed was removed and thecontainers cleaned if necessary. When the weight of the larvae wasmeasured the containers were emptied and thoroughly cleaned. The baseweight for 30 larvae in each container was determined prior to the startof the test feed. Three parallel containers were given feed with eachconcentration of alginate and three containers were given the controlfeed. Feed was given several times during the daytime (approximatelyevery hour to ensure feed was not a limiting factor.

Every 10 days the weight of 30 larvae was measured. The experimentlasted for 60 days. The specific growth rate was calculated as inExample 5. The specific growth rates are given in FIG. 10. Bars markedwith an “*” indicates a significant difference (p<0.05).

EXAMPLE 9

The mortality in the growth study of spotted wolffish larvae in example7 was registered and is illustrated in FIG. 11. No significantdifference between any of the groups was measured (p<0.05).

EXAMPLE 10

The mortality in the growth study of cod larvae in example 8 wasregistered and is illustrated in FIG. 12. No significant differencebetween any of the groups was measured (p<0.05).

1. An oral, immunostimulating material for fish comprising animmunostimulating amount of an alginate having a mannuronic acid (M)content of at least 40% and an acceptable carrier.
 2. The material ofclaim 1 wherein said M content is 50% to 70%.
 3. The material of claim 1wherein said M content is 70% to 80%.
 4. The material of claim 1 whereinsaid M content is 80% to 99.9%.
 5. The material of claim 2 wherein saidalginate is derived from Lessonia seaweed, Durvillea seaweed, orLaminaria seaweed.
 6. The material of claim 3 wherein said alginate isderived from Durvillea seaweed.
 7. The material of claim 4 wherein saidalginate is derived from bacteria or Durvillea water extract.
 8. Thematerial of claim 1, wherein said material is (i) a pharmaceutical,veterinary or nutraceutical solid dosage form or liquid formulation,(ii) a solid, semi-solid or liquid food or (iii) a solid or liquidnutritional supplement.
 9. A method of stimulating the immune system ofa fish comprising orally administering an immunostimulating amount of animmunostimulating material to said fish, wherein said material comprisesan alginate having a M content of at least 40% and an acceptablecarrier.
 10. The method of claim 9 wherein said M content is 50% to 70%.11. The method of claim 9 wherein said M content is 70% to 80%.
 12. Themethod of claim 9 wherein said M content is 80% to 99.9%.
 13. The methodof claim 9, wherein said ingestible material is (i) a pharmaceutical,veterinary or nutraceutical solid dosage form or liquid formulation,(ii) a solid or liquid food or (iii) a solid or liquid nutritionalsupplement.
 14. An oral, immunostimulating material for fish comprisingan immunostimulating amount of an alginate having a M content of atleast 80% and an acceptable carrier, wherein said alginate is made bymixing and swelling Durvillea seaweed in water, maintaining the pH aboveabout 2.3 at a temperature above 20° C. for at least 30 minutes,isolating a solubilised fraction from the mixture and recovering saidalginate having a M content of at least 80% from the solubilizedfraction by precipitation with acid, salt or alcohol.
 15. The materialof claim 14, wherein said material is (i) a pharmaceutical, veterinaryor nutraceutical solid dosage form or liquid formulation, (ii) a solidor liquid food or (iii) a solid or liquid nutritional supplement.
 16. Amethod of stimulating the immune system of a fish comprising orallyadministering an immunostimulating amount of the immunostimulatingmaterial of claim 14 to said mammal, birds, and reptile.
 17. A methodfor stimulating weight gain in fish through immunostimulation comprisingorally administering an immunostimulating amount of an immunostimulatingmaterial to said fish, wherein said material comprises an alginatehaving a M content of at least 40% and an acceptable carrier.
 18. Themethod of claim 17 wherein said M content is 50% to 70%.
 19. The methodof claim 17 wherein said M content is 70% to 80%.
 20. The method ofclaim 17 wherein said M content is 80% to 99.9%.
 21. An oral,immunostimulating material for fish comprising an immunostimulatingamount of an alginate having a M content of at least 40%.
 22. Thematerial of claim 21, wherein said M content is 50 to 70%.
 23. Thematerial of claim 21, wherein said M content is 70 to 80%.
 24. Thematerial of claim 21, wherein said M content is 80 to 99.9%.
 25. Amethod of stimulating the immune system of a fish comprising orallyadministering an immunostimulating amount of an immunostimulatingmaterial to said fish, wherein said material comprises an alginatehaving a M content of at least 40%.
 26. The method of claim 25, whereinsaid M content is 50% to 70%.
 27. The method of claim 25, wherein said Mcontent is 70% to 80%.
 28. The method of claim 25, wherein said Mcontent is 80% to 99.9%.
 29. An oral, immunostimulating material forfish comprising an immunostimulating amount of an alginate having a Mcontent of at least 40%, with the proviso that said alginate is eithersynthetically made or isolated from a seaweed or bacterial source. 30.The material of claim 29, wherein said M content is 50 to 70%.
 31. Thematerial of claim 29, wherein said M content is 70 to 80%.
 32. Thematerial of claim 29, wherein said M content is 80 to 99.9%.
 33. Thematerial of claim 29, further comprising an acceptable carrier.
 34. Thematerial of claim 29, wherein said material is (i) a pharmaceutical,veterinary or nutraceutical solid dosage form or liquid formulation,(ii) a solid, semi-solid or liquid food or (iii) a solid or liquidnutritional supplement.
 35. A method of stimulating the immune system ofa fish comprising orally administering an immunostimulating amount of animmunostimulating material to said fish, wherein said material comprisesan alginate having a M content of at least 40% with the proviso thatsaid alginate is either synthetically made or isolated from a seaweed orbacterial source.
 36. The method of claim 35 wherein said materialfurther comprises an acceptable carrier.
 37. The method of claim 35,wherein said M content is 50 to 70%.
 38. The method of claim 35, whereinsaid M content is 70 to 80%. 39 The method of claim 35, wherein said Mcontent is 80 to 99.9%. 40 The method of claim 35, further comprising anacceptable carrier.
 41. The method of claim 35, wherein said material is(i) a pharmaceutical, veterinary or nutraceutical solid dosage form orliquid formulation, (ii) a solid, semi-solid or liquid food or (iii) asolid or liquid nutritional supplement.